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Genetically-encoded Forster resonance energy transfer (FRET) biosensors enable us to visualize a variety of signaling events, such as protein phosphorylation and G protein activation in living cells (Miyawaki, 2003). Using FRET-based biosensors we can obtain spatiotemporal information on the changes in activity of signaling molecules in living cells. From this viewpoint, FRET imaging of signaling molecules that regulate membrane traffic is one of the most suitable applications of this technique. The Rab family GTPases constitute the largest branch of the Ras GTPase superfamily. Rab GTPases use the guanine nucleotidedependent switch mechanism common to the Ras superfamily to regulate each of the four major steps in membrane trafficking: vesicle budding, vesicle delivery, vesicle tethering, and fusion of the vesicle membrane with that of the target compartment (Zerial and McBride, 2001; Grosshans et al., 2006; Stenmark, 2009). Recently, we developed a FRET sensor for Rab5, and demonstrated that live-cell imaging with FRET sensors enables us to pinpoint the activation and inactivation of Rab5, and thereby to understand its relationship with other events linked to vesicle transport (Kitano et al., 2008). In the first half of this chapter, we describe step-by-step strategies to develop unimoleculartype FRET biosensors for Rab family GTPases. We use the development of a Rab35 sensor as an example. Although improvements to FRET sensors are still made on a trial-and-error basis, we provide practical tips for their optimization. In the second half of this chapter, we introduce FRET imaging with total internal reflection fluorescence (TIRF) microscopy. TIRF microscopy is particularly well suited to visualize the dynamics of molecules and events near the plasma membrane (Mattheyses et al., 2010). We have used FRET imaging with TIRF microscopy to show that the activity of TC10, a Rho family GTPase, at tethered vesicles drops immediately before vesicle fusion in HeLa cells stimulated with epidermal growth factor (EGF) (Kawase et al., 2006). We describe how to set up and use TIRF-FRET to visualize local changes in GTPase activity on vesicles during membrane fusion.

How to make FRET biosensors for Rab family GTPases